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Maize transformation method

A technology of corn and callus, applied in horticultural methods, botanical equipment and methods, using vectors to introduce foreign genetic material, etc., can solve the problems of poor stability and low conversion rate, and achieve cost-saving and easy-to-obtain effects

Inactive Publication Date: 2013-11-27
BEIJING DABEINONG TECH GRP CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] The purpose of the present invention is to provide a method for transforming corn, which can effectively overcome the technical defects of the prior art such as seasonality, poor stability, serious tissue characteristics, and low conversion rate.

Method used

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  • Maize transformation method
  • Maize transformation method

Examples

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Effect test

no. 1 example

[0043] The first embodiment, construction of recombinant expression vector and transformation of recombinant expression vector into Agrobacterium

[0044] 1. Construct a recombinant cloning vector containing the target gene

[0045] The Cry1Ab nucleotide sequence was connected to the cloning vector pGEM-T (Promega, Madison, USA, CAT: A3600), and the operation steps were carried out according to the instructions of the pGEM-T vector produced by Promega Company to obtain the recombinant cloning vector DBN01Ab-T, and its construction process Such as figure 1Shown (wherein, Amp represents the ampicillin resistance gene; f1 represents the replication origin of phage f1; LacZ is the LacZ start codon; SP6 is the SP6 RNA polymerase promoter; T7 is the T7 RNA polymerase promoter; Cry1Ab is the Cry1Ab nucleoside acid sequence (SEQ ID NO: 1); MCS is a multiple cloning site).

[0046] Then, the recombinant cloning vector DBN01Ab-T was transformed into Escherichia coli T1 competent cells...

no. 2 example

[0053] The second embodiment, the acquisition of corn plants transferred to the Cry1Ab nucleotide sequence

[0054] Stripping of corn immature embryos: Sow the seeds of corn variety Zong 31 (Z31) in the greenhouse or field, wait for the corn plants to grow 7-10 days after pollination, when the length of the immature embryos is 0.6-1mm, the maximum length is 1mm. Good, take the immature embryos, in the induction medium (MS salt 4.3g / L, MS vitamins, sucrose 30g / L, L-proline 0.7g / L, 2,4-dichlorophenoxyacetic acid (2,4 -D) The callus was induced on 1.5mg / L, silver nitrate 0.85mg / L, 2-morpholineethanesulfonic acid (MES) 0.5g / L), and the induction time was 2 weeks. Transfer the induced calli (500) to the pre-culture medium (MS salt 4.3g / L, MS vitamin, sucrose 30g / L, L-proline 0.7g / L, silver nitrate 0.85mg / L, 2-morpholineethanesulfonic acid (MES) 0.5g / L, 2,4-dichlorophenoxyacetic acid (2,4-D) 1.5mg / L) for pre-culture, the pre-culture time is 4-7 days . Subculture the callus that h...

no. 3 example

[0061] The third embodiment, using TaqMan to verify the corn plant transferred to the Cry1Ab nucleotide sequence

[0062]About 100mg of the leaves of the maize plant transformed with the Cry1Ab nucleotide sequence was taken as a sample, and its genomic DNA was extracted with Qiagen's DNeasy Plant Maxi Kit, and the copy number of the Cry1Ab gene was detected by the fluorescent quantitative PCR method of Taqman probe. At the same time, wild-type maize plants were used as a control, and detection and analysis were carried out according to the above method. The experiment was repeated 3 times, and the average value was taken.

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Abstract

The invention relates to a maize transformation method which comprises the following steps: stripping young maize embryos for inducing callus; and infecting the callus by agrobacterium. The maize transformation method fulfills the purpose that the callus induced by the young maize embryos serves as an acceptor material for transformation for the first time, and ensures that the transformation rate can reach up to 5%; meanwhile, the maize callus with high quality and large amount can be obtained for subculture propagation, and can be easily available and also be supplied on a large scale, so that the requirement of maize transformation on the acceptor material can be fully met; the defect that the harvest condition of explants cannot be accurately predicted due to the limitation of weather conditions on field materials can be overcome, so that maize genetic transformation is not influenced by seasons, and the requirement on annual stable transformation can be met; in addition, the dependence on the young maize embryo in the prior art is cut, and maize does not need to be planted in a greenhouse and the like in non-maize-planting seasons, so that the cost for obtaining the acceptor material is greatly reduced.

Description

technical field [0001] The invention relates to a method for plant transformation, in particular to a method for transforming corn callus. Background technique [0002] Corn is one of the three major food crops in the world, and it is also an important industrial raw material and bioenergy material. It plays a pivotal role in the food production of our country and the world. Among the many factors that increase the yield of corn, the role of new variety selection accounts for 40%. With the rapid development of molecular biology technology, transgenic technology has begun to play an important role in the breeding of new maize varieties. GM corn has become the second largest GM crop in the world after GM soybean. [0003] A good receptor system is an important part of maize genetic transformation, which is related to the provision of suitable receptor materials for transformation and whether the transformed cells can regenerate into normal plants. In the receptor system, ho...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82A01H4/00A01H5/00
Inventor 贾志伟
Owner BEIJING DABEINONG TECH GRP CO LTD
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